Protein Kinase A) Activity Kit

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WEB INSERT 120517

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DetectX®

PKA (Protein Kinase A) Activity Kit

Catalog Number K027-H1

Sp e c i e s In d e p e n d e n t

Sample Types Validated:

Cell Lysate, Tissue Extracts and Buffer Samples

Please read this insert completely prior to using the product.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

New! DualRead™ Assay Extended Standard Curve Range

1 WEB INSERT 120517 Ta b l e Of Co n t e n t s

Background 3

Assay Principle 4

Related Products 4

Supplied Components 5

Storage Instructions 5

Other Materials Required 6

Precautions 6

Sample Types 7

Sample Preparation 7

Reagent Preparation 8

Assay Protocol 9

Calculation of Results 10

Typical Data 11

Validation Data Sensitivity, Linearity, etc. 12

Inhibition Studies 13

Interferents and Cross Reactivity 14

Warranty & Contact Information 15

Plate Layout Sheet 16

www.ArborAssays.com 2 WEB INSERT 120517 Ba c k g r o u n d

PKA was discovered in the laboratory of Edwin G. Krebs in the 1960’s1. This important class of kinases, refered to as Arg-directed kinases or AGC-family kinases, includes cAMP-dependent protein kinase (PKA or cAPK), cGMP-dependent protein kinase (PKG), protein kinase C, Akt and RSK. These kinases share a substrate specificity characterized by Arg at position 3 relative to the phosphorylated serine or threonine2-4. The second messenger cyclic AMP (cAMP) activates PKA in mammalian cells and controls many cellular mechanisms such as gene transcription, ion transport, and protein phosphorylation2. Inactive PKA is a heterotetramer composed of a regulatory subunit (R) dimer and a catalytic subunit (C) dimer. In this inactive state, the pseudosubstrate sequences on the R subunits block the active sites on the C subunits. PKA shares substrate specificity with Akt (PKB) and PKC4. Substrates that present this consensus sequence and are phosphorylated by PKA are Bad (Ser155), CREB (Ser133), and GSK-3 (GSK-3α Ser21 and GSK-3ß Ser9) 5-7.

PKA has been implicated in numerous cellular processes, including modulation of other protein kinases, regulation of intracellular calcium concentration, and regulation of transcription8. Tran- scriptional responses to increased cAMP occur through activation of the cAMP response element– binding protein (CREB), cAMP response element modulator (CREM), and activating transcription factor 1 (ATF1) 9. Each of these transcription factors contains a kinase-inducible domain containing a conserved site for phosphorylation by PKA.

1. Walsh, DA, Perkins, JP, Krebs, EG., “An adenosine 3’,5’-monophosphate-dependent protein kinase from rabbit skeletal muscle.”, J. Biol. Chem. 1968, 243:3763–3765. 2 Montminy, M. “Transcriptional regulation by cyclic AMP. “ Annu Rev Biochem., 1997, 66:807-822. 3. Pearson, RB. and Kemp, BE. “Protein kinase phosphorylation site sequences and consensus specificity motifs: Tabulations “., Methods Enzymol., 1991, 200:62-81. 4. Dell’Acqua, ML. and Scott, JD. “Protein kinase A anchoring. “, J. Biol. Chem., 1997, 272:12881-12884. 5. Tan, Y. et al. “BAD Ser-155 Phosphorylation Regulates BAD/Bcl-XL Interaction and Cell Survival “, J. Biol. Chem., 2000, 275:25865-25869. 6. Gonzalez, GA. and Montminy, MR. “Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. “, Cell, 1989, 59:675-680. 7. Fang, X. et al. “Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A”., Proc. Natl. Acad. Sci. USA 2000, 97:11960-11965. 8. Taskén, K, and Aandahl, EM., “Localized effects of cAMP mediated by distinct routes of protein kinase A.”., Physiol. Rev., 2004, 84:137–167. 9. Sands, WA, and Palmer, TM., “Regulating gene transcription in response to cAMP elevation.”, Cell. Signal. 2008, 20:460–466.

www.ArborAssays.com 3 WEB INSERT 120517 Ass a y Pr i n c i p l e The DetectX® PKA (Protein Kinase A) Activity kit is designed to quantitatively measure PKA activity in a variety of samples. Please read the complete kit insert before performing this assay. A recombinant PKA standard is provided to generate a standard curve for the assay and all samples should be read off the standard curve. For samples that may have high PKA activity we offer an alternative protocol that reads the substrate reaction at 650 nm. The kit utilizes an immobilized PKA substrate bound to a microtiter plate. Samples containing PKA will, in the presence of the supplied ATP, phosphorylate the immobilized PKA substrate. A rabbit antibody specific for the phospho-PKA substrate binds to the modified immobilized substrate. An antibody specific for rabbit IgG labeled with peroxidase is then added to the plate to bind to the rabbit anti-phospho- PKA substrate. After a short incubation and wash, substrate is added and the intensity of the color developed is directly proportional to the amount of PKA in the samples and standards.

PKA Containing ATP REACTION OVERVIEW Sample

GLOSSARY PO PO 4 4

PKA Substrate

PO Phospho PKA Substrate 4 Antibody

Phospho-PKA Substrate HRP HRP Goat anti Rabb t IgG HRP HRP

PO PO 4 PO 4 PO 4 4

TMB Substrate HRP HRP

PO PO 4 READ 4

Related Products

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www.ArborAssays.com 4 WEB INSERT 120517 Su p p l i e d Co m p o n e n t s

PKA Substrate 96 Well Plate 1 Plate Catalog Number C107-1EA Break-apart strip microtiter plate coated with PKA Substrate

PKA Standard 2 vials Catalog Number C131-1EA 5,000 Units of recombinant fully active PKA in special stabilizing buffer. One unit is defined as the amount of PKA required to catalyze the transfer of 1 pmol of ATP phos- phate to substrate in 1 minute at 30°C. PKA Standard must be stored at -20°C.

ATP 1 vial Catalog Number X103-1EA ATP lyophilized. Store in supplied desiccator.

Phospho PKA Substrate Antibody 3 mL Catalog Number C104-3ML A solution of rabbit antibody specific for phospho-Substrate.

Goat anti-Rabbit IgG HRP Conjugate 3 mL Catalog Number C105-3ML A solution of goat antibody specific for rabbit IgG labeled with peroxidase.

Kinase Reaction Buffer Concentrate 60 mL Catalog Number X104-60ML A 2X concentrate containing detergents and stabilizers.

Cell Lysis Buffer 100 mL Catalog Number X050-100ML A Tris based buffer containing detergents. Store Frozen as this buffer contains no preservatives Wash Buffer Concentrate 30 mL Catalog Number X007-30ML A 20X concentrate that should be diluted with deionized or distilled water. TMB Substrate 11 mL Catalog Number X019-11ML

Stop Solution 5 mL Catalog Number X020-5ML 1M solution of hydrochloric acid. CAUSTIC. Plate Sealer 2 Each Catalog Number X002-1EA

St o r a g e In s t r u c t i o n s

The PKA Standard and Cell Lysis Buffer must be stored at -20°C. The Cell Lysis Buffer has no pre- servative and must be kept frozen at -20°C. All components of this kit can be stored together at -20°C. The kit must be used prior to the expiration date on the kit box label.

www.ArborAssays.com 5 WEB INSERT 120517 Ot h e r Ma t e r i a l s Re q u i r e d Distilled or deionized water. Glass test tubes. Shaking plate incubator capable of maintaining 30°C. Repeater pipet and disposable tips capable of dispensing 10, 25, 50 and 100 µL accurately. The following Protease inhibitors must be added to all buffers that are used to measure PKA activity. See pages 7 & 8. • Phenylmethanesulfonyl fluoride (PSMF), such as Sigma 78830. • A universal protease inhibitor cocktail (PIC) such as Sigma P1860 or Roche 05892970001. In addition: • The phosphatase inhibitor, Sodium Orthovanadate, must be activated and added to the Cell Lysis buffer. See activation instruction opposite. Colorimetric 96 well microplate reader capable of reading optical density at 450 and 650 nm. Software for converting raw relative optical density readings from the plate reader and carrying out four parameter logistic curve (4PLC) fitting. Contact your plate reader manufacturer for details. DualRead™ System This kit uses our unique DualRead™ system. We include instructions for an alternative high standard which would typically generate ODs at 450 nm too high to be read on most plate readers. By reading the plate at 650 nm (where TMB optical density is about 3 fold lower) immediately before addition of the Stop Solution some samples outside the normal standard curve range can be read. See instructions on pages 8-10.

Pr e c a u t i o n s

The PKA Standard and Cell Lysis Buffer MUST be stored at -20°C.

As with all such products, this kit should only be used by qualified personnel who have had laboratory safety instruction. The complete insert should be read and understood before attempting to use the product.

This kit utilizes a peroxidase-based readout system. Buffers, including other manufacturers Wash Buffers, containing sodium azide will inhibit color production from the enzyme. Make sure all buffers used for samples are azide free. Ensure that any plate washing system is rinsed well with deionized water prior to using the supplied Wash Buffer as prepared on Page 8.

The Stop Solution is acid. The solution should not come in contact with skin or eyes. Take appropriate precautions when handling this reagent.

www.ArborAssays.com 6 WEB INSERT 120517 Sa m p l e Ty p e s This assay has been validated for Jurkat cell lysates. Samples containing visible particulate should be centrifuged prior to using.

Sa m p l e Pr e p a r a t i o n Cells must be lysed in the Activated Cell Lysis Buffer, after addition of protease inhibitors and activated orthovanadate to the provided Cell Lysis Buffer (see below). All cells and the lysates made from them must be stored at ≤ -70°C and should be stored as aliquots for single use. Do not freeze-thaw samples. Do not store cells or lysates above -70°C. The preparation of Activated Sodium Orthovanadate is as follows: Preparation of Activated Orthovanadate 200 mM Activated Orthovanadate should be prepared by dissolving 1.84 g of sodium orthovanadate in 45 mL of water. Adjust the pH of the solution to 10 with 1M NaOH or HCl. At pH 10 the solution should be yellow. Boil the solution until it turns colorless (approximately 10 min). All of the orthovanadate should dissolve. Cool to room temperature and readjust the pH to 10. Repeat the boiling of the solution and pH readjustment until the solution is colorless and remains at pH 10. Adjust the final volume to 50 mL with water. Store the Activated Sodium Orthovanadate in aliquots and freeze at -20°C. Use an aliquot for preparing Activated Cell Lysis Buffer and discard.

Preparation of Activated Cell Lysis Buffer Prepare the Activated Cell Lysis Buffer by addition of 1 µL of PIC per mL of Cell Lysate Buffer. Add 1 mM PMSF and 10 mM Activated Orthovanadate. The resulting Activated Cell Lysis Buffer is a pH 8 Tris based buffer containing 1% NP-40 as a cell disruption agent. This assay may not be compatible with other cell lysis buffers containing high concentrations of SDS or other detergents and erroneous activity measurements may result.

Cell Lysis PKA Activity: Jurkat Cell Lysate Dilution 18 Add prepared Activated Cell Lysis Buffer to the cells (for Jurkat cells, we lysed at 100 million cells per mL). 16 Incubate for 30 minutes on ice with occasional vor- 14 texing. Centrifuge at 10,000 rpm at 4°C for 10 min- 12 utes and carefully aspirate off the supernatant for 10 analysis. Supernatants can be frozen at ≤ -70°C for later analysis. 8 6

The supernatants should be diluted at least 1:10 into (U/mL) Activity PKA 4 prepared Ki n a s e Ass a y Bu f f e r (see Page 8) prior to 2 running in the assay. It is recommended that a con- 0 trol lysate be serially diluted in Ki n a s e Ass a y Bu f f e r to 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 determine the appropriate dilution to obtain a linear Jurkat Cell Lysate Diln (x 10^6)/mL response. See Graph at right.

Samples diluted in Ki n a s e Ass a y Bu f f e r can be frozen at ≤ -70°C for analysis later. ®

www.ArborAssays.com 7 WEB INSERT 120517 Re a g e n t Pr e p a r a t i o n Allow the kit reagents to come to room temperature for 30 minutes, except for the standard which must be kept on ice. We recommend that all standards and samples be run in duplicate to allow the end user to accurately determine PKA activity. Keep all samples on ice and ensure they have been diluted appropriately prior to running them in the kit.

Ki n a s e Ass a y Bu f f e r - CRITICAL STEP! Dilute Kinase Reaction Buffer Concentrate 1:2 by adding one part of the concentrate to one part of deionized water. Add 0.5 µL/mL of PIC and PMSF to 1 mM to make Ki n a s e Ass a y Bu f f e r . Use within 8 hours. ATP Prior to opening vial, tap vial on bench top to ensure contents are in base of vial. Add 1.2 mL of prepared Ki n a s e Ass a y Bu f f e r to the ATP vial. Vortex to solubilize. Once diluted, store any unused ATP solution at -20°C for up to 3 months. Wash Buffer Dilute Wash Buffer Concentrate 1:20 by adding one part of the concentrate to nineteen parts of deionized water. Once diluted this is stable at room temperature for 3 months. Standard Preparation Spin down the contents of the PKA Standard vial in a microcentrifuge for 2 minutes at 14,000 rpm at 4°C. Keep all standards on ice during use.

Prepare an Intermediate Stock dilution by pipetting 1 mL of prepared Ki n a s e Ass a y Bu f f e r into the PKA standard vial. Invert vial and vortex thoroughly to ensure complete mixing of contents. This Intermediate Stock will have an activity of 5,000 Units/mL. Label five tubes as #1 through #5. Pipet the standards using the Intermediate Stock according to the table below. The activity of PKA in tubes 1 through 5 will be 25, 20, 15, 10, and 5 Units/mL. Alternative High Standard For samples that may exceed 25 U/mL prepare the 40 U/mL Alterna- tive Standard by pipetting 8 µL of Intermediate Stock into 992 µL of prepared Ki n a s e Ass a y Bu f f e r .

Keep all Standards on Ice and use within 30 minutes of preparation.

Standard Activity, U/mL Std 1 Std 2 Std 3 Std 4 Std 5 (25 U/mL) (20 U/mL) (15 U/mL) (10 U/mL) (5 U/mL)

Ki n a s e Ass a y Bu f f e r (µL) 995 60 60 60 60 Addition 5 µL Intermediate 240 µL 180 µL 120 µL 60 µL Stock Std 1 Std 2 Std 3 Std 4

www.ArborAssays.com 8 WEB INSERT 120517 Ass a y Pr o t o c o l

1. Use the plate layout sheet on the back page of the insert to aid in proper sample and standard identification.

2. Pipet 40 µL of Ki n a s e Ass a y Bu f f e r as a Zero Standard, and samples or standards diluted in Ki n a s e Ass a y Bu f f e r into duplicate wells in the plate.

3. Add 10 µL of the reconstituted ATP to each of the wells using a repeater pipet.

4. Seal the plate and incubate at 30°C shaking for 90 minutes.

5. Aspirate the plate and wash each well 4 times with 300 µL wash buffer. Tap the plate dry on clean absorbent towels.

6. Add 25 µL of the Goat anti-rabbit IgG HRP conjugate to each well using a repeater pipet.

7. Add 25 µL of the Rabbit Phospho PKA Substrate antibody to each well using a repeater pipet.

8. Seal the plate and incubate the plate at room temperature for 60 minutes with shaking.

9. Aspirate the plate and wash each well 4 times with 300 µL wash buffer. Tap the plate dry on clean absorbent towels.

10. Add 100 µL of the TMB Substrate Solution to each well, using a repeater pipet.

11. Incubate the plate at room temperature for 30 minutes.

DualRead™ If the blue substrate color of any of your samples appears darker than the 25 U/mL standard we recommend reading the plate at 650 nm, immediately prior to adding stop solution.

12. Add 50 µL of the Stop Solution to each well, using a repeater pipet.

13. Read the optical density generated from each well in a plate reader capable of reading at 450 nm.

14. Use the plate reader’s built-in 4PLC software capabilities to calculate PKA activity for each sample.

www.ArborAssays.com 9 WEB INSERT 120517 Ca l c u l a t i o n o f Re s u l t s Average the duplicate 450 nm (and optional 650 nm) OD readings for each standard and sample. Create a standard curve by reducing the data using the 4PLC fitting routine on the plate reader, after subtracting the mean ODs for the zero standard. The sample activity obtained should be multiplied by the dilution factor to obtain neat sample values.

Ty p i c a l Da t a (450 n m Re a d ) Sample Mean OD Net OD PKA Activity (450nm) (450nm) (U/mL) Standard 1 1.272 1.223 25 Standard 2 0.929 0.880 20 Standard 3 0.618 0.569 15 Standard 4 0.363 0.314 10 Standard 5 0.155 0.106 5 Zero 0.049 0.000 0 Sample 1 0.158 0.109 5.91 Sample 2 1.000 0.951 20.9

Ty p i c a l d a t a (650 n m Re a d ) Sample Mean OD Net OD PKA Activity (650nm) (650nm) (U/mL) Alt. Std. 0.564 0.528 40 Standard 1 0.42 0.384 25 Standard 2 0.307 0.271 20 Standard 3 0.23 0.195 15 Standard 4 0.102 0.066 10 Standard 5 0.06 0.025 5 Zero 0.036 0 0 Sample 1 0.274 0.238 18.8 Sample 2 0.109 0.073 7.86

Always run your own standard curve for calculation of results. ® Do not use this data.

www.ArborAssays.com 10 WEB INSERT 120517 Typical Standard Curves

450 nm Read

1.4

1.2

1.0

0.8 (450nm)

0.6 4 Net OD

0.4

0.2

0.0 0 5 10 15 20 25 PKA Activity (U/mL)

650 nm Read

0.6

0.5

0.4

0.3 (650nm)

0.2 Net OD

0.1

0 0 5 10 15 20 25 30 35 40 PKA Activity (U/mL) ®

www.ArborAssays.com 11 WEB INSERT 120517 Va l i dat i o n Da t a

Sensitivity Sensitivity was calculated by comparing the OD’s for twenty wells run for each of the zero and the 5 Unit/mL standard. The detection limit was determined at two (2) standard deviations from the zero along the standard curve. Sensitivity was determined as 0.366 Units/mL. This is equivalent to 14.6 milli Units/sample.

Linearity Linearity was determined by taking two Jurkat cell lysate samples, one with a high PKA activity and one with a lower PKA activity , and mixing in the ratios given below. The measured activities were compared to the expected values based on the ratios used.

Low High Observed Expected % Recovery Sample Sample Activity (mU/mL) Activity (mU/mL) 0% 100% 8.88 ------20% 80% 7.96 8.00 99.5% 40% 60% 7.39 7.12 103.7% 60% 40% 6.41 6.24 102.6% 80% 20% 5.41 5.37 100.8% 100% 0% 4.49 ------Mean Recovery 101.6%

9.0

y = 1.0021x - 0.1212 8.0 R2 = 0.9858

7 . 0

Expected Activity (U/mL) Activity Expected 6.0

5.0 5.0 6.0 7 . 0 8.0 9.0 Observed Activity (U/mL)

www.ArborAssays.com 12 WEB INSERT 120517 In h i b i t i o n St u d i e s

Studies with recombinant PKA Approximately 30 Units/mL of human recombinant PKA was incubated with H 89 dihydrochloride from 0 to 10,000 nM in Assay Buffer for 30 minutes at room temperature prior to running in the assay. % Activity is expressed by comparison to the activity of the buffer control (28.02 U/mL). 4PLC data comparison determined the IC50% to be 19.1 nM.

100

A60 tiv % Activity

30 0 10 100 1,000 10,000 H 89 Conc. (nM)

Studies with Cell Lysates Aliquots of a Jurkat cell lysate containing approximately 40,000 cells were treated with the re- versible PKA inhibitor H 89 dihydrochloride or Assay Buffer as the control and run in the assay.

6 PKA Activity (U/mL) Activity PKA

0 + H 89 PKA Inhibitor Control Reaction Condition

www.ArborAssays.com 13 WEB INSERT 120517 In t e r f e r e n t s

A variety of solvents were tested as possible interfering substances in the assay. 0.5% ethanol in the well decreased the activity recorded by 12.7%, whereas 0.10% ethanol in the well decreased activity by 3.7%. 0.5% DMSO in the well decreased activity by 2.8%. 0.1% methanol in the well increased activity by 3.1%. We expect solvent levels at 0.1% of well volume to have little or no effect on the measured activity. A solvent only control should be run by the end user when appropriate.

Cr oss Re a c t i v i t y

Protein Kinase % Cross Reactivity PKAc alpha 100% PKAc beta 73% PKAc gamma 10.1%

www.ArborAssays.com 14 WEB INSERT 120517 Li m i t e d Wa r r a n t y

Arbor Assays warrants that at the time of shipment this product is free from defects in materials and workmanship. This warranty is in lieu of any other warranty expressed or implied, including but not limited to, any implied warranty of merchantability or fitness for a particular purpose.

We must be notified of any breach of this warranty within 48 hours of receipt of the product. No claim shall be honored if we are not notified within this time period, or if the product has been stored in any way other than outlined in this publication. The sole and exclusive remedy of the customer for any liability based upon this warranty is limited to the replacement of the product, or refund of the invoice price of the goods.

Co n t a c t In f o r m a t i o n

For details concerning this kit or to order any of our products please contact us:

Arbor Assays 1514 Eisenhower Place Ann Arbor, Michigan 48108 USA Phone: 734-677-1774 Fax: 734-677-6860 Web: www.ArborAssays.com

E Mail Addresses: [email protected] [email protected] [email protected] [email protected]

DetectX®, ThioStar® and the Arbor Assays logo are all registered trademarks.

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WEB INSERT 120517

G C B H F E D A 1 2 3 4 5 6 7 8 9 10 11 12 1

K027-H1 16 17May12

K015-F.2